A Novel Leishmania major Amastigote Assay in 96-Well Format for Rapid Drug Screening and Its Use for Discovery and Evaluation of a New Class of Leishmanicidal Quinolinium Salts

Abstract

bIn most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC 50 ). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC 50 s against intracellular amastigotes (IC 50 < 1 g/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC 50 ‫؍‬ 0.03 g/ml; SI ‫؍‬ 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity.

Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester

“…Compounds 1, 2, 7, 10, 15, 19, 20 showing high activity against L. major promastigotes were selected for screening against L. major amastigotes and cytotoxicity against BMDM. The amastigote assay was conducted as reported by Bringmann et al [16] and the results are presented in Table 4. The cytotoxicity against BMDM mirrors those against the macrophages J774.1.…”

“…Antileishmanial activities of the synthesized compounds were evaluated in L. major promastigotes as described before [16] and in L. donovani promastigotes according to Hazra et al [17]. Cytotoxicity was determined on a J774.1 murine cell line [16].…”

“…Cytotoxicity was determined on a J774.1 murine cell line [16]. The antiprotozoal activities are presented in Tables 1-3 as inhibitory concentrations (IC 50 ).…”

“…AlamarBlue assays for investigation of antileishmanial activities against L. major promastigotes, amastigotes and J774.1 and BMDM cytotoxicity were conducted as previously reported [16]. The protocol for the L. donovani quantitative colorimetric assay is outlined by Hazra et al [17].…”

Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl and Thymyl Cinnamate Induce Cytoplasmic Vacuoles and Death in Schistosomula of Schistosoma mansoni

“…Compounds 18, 28 and 29 were synthesized via Steglich esterification as explained there. The protocol for the cytotoxicity studies against J774.1 was as described [35]. …”